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Chemical proteomics tracks virus entry and uncovers NCAM1 as Zika virus receptor.

ABSTRACT: The outbreak of Zika virus (ZIKV) in 2016 created worldwide health emergency which demand urgent research efforts on understanding the virus biology and developing therapeutic strategies. Here, we present a time-resolved chemical proteomic strategy to track the early-stage entry of ZIKV into host cells. ZIKV was labeled on its surface with a chemical probe, which carries a photocrosslinker to covalently link virus-interacting proteins in living cells on UV exposure at different time points, and a biotin tag for subsequent enrichment and mass spectrometric identification of the receptor or other host proteins critical for virus internalization. We identified Neural Cell Adhesion Molecule (NCAM1) as a potential ZIKV receptor and further validated it through overexpression, knockout, and inhibition of NCAM1 in Vero cells and human glioblastoma cells U-251 MG. Collectively, the strategy can serve as a universal tool to map virus entry pathways and uncover key interacting proteins.

SUBMITTER: Srivastava M

PROVIDER: S-EPMC7403387 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Chemical proteomics tracks virus entry and uncovers NCAM1 as Zika virus receptor.

Srivastava Mayank M Zhang Ying Y Chen Jian J Sirohi Devika D Miller Andrew A Zhang Yang Y Chen Zhilu Z Lu Haojie H Xu Jianqing J Kuhn Richard J RJ Andy Tao W W

Nature communications 20200804 1

The outbreak of Zika virus (ZIKV) in 2016 created worldwide health emergency which demand urgent research efforts on understanding the virus biology and developing therapeutic strategies. Here, we present a time-resolved chemical proteomic strategy to track the early-stage entry of ZIKV into host cells. ZIKV was labeled on its surface with a chemical probe, which carries a photocrosslinker to covalently link virus-interacting proteins in living cells on UV exposure at different time points, and ...[more]

PMID: 32753727

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Project description:BackgroundThe flaviviridae family comprises single-stranded RNA viruses that enter cells via clathrin-mediated pH-dependent endocytosis. Although the initial events of the virus entry have been already identified, data regarding intracellular virus trafficking and delivery to the replication site are limited. The purpose of this study was to map the transport route of Zika virus and to identify the fusion site within the endosomal compartment.MethodsTracking of viral particles in the cell was carried out with confocal microscopy. Immunostaining of two structural proteins of Zika virus enabled precise mapping of the route of the ribonucleocapsid and the envelope and, consequently, mapping the fusion site in the endosomal compartment. The results were verified using RNAi silencing and chemical inhibitors.ResultsAfter endocytic internalization, Zika virus is trafficked through the endosomal compartment to fuse in late endosomes. Inhibition of endosome acidification using bafilomycin A1 hampers the infection, as the fusion is inhibited; instead, the virus is transported to late compartments where it undergoes proteolytic degradation. The degradation products are ejected from the cell via slow recycling vesicles. Surprisingly, NH4Cl, which is also believed to block endosome acidification, shows a very different mode of action. In the presence of this basic compound, the endocytic hub is reprogrammed. Zika virus-containing vesicles never reach the late stage, but are rapidly trafficked to the plasma membrane via a fast recycling pathway after the clathrin-mediated endocytosis. Further, we also noted that, similarly as other members of the flaviviridae family, Zika virus undergoes furin- or furin-like-dependent activation during late steps of infection, while serine or cysteine proteases are not required for Zika virus maturation or entry.ConclusionsZika virus fusion occurs in late endosomes and is pH-dependent. These results broaden our understanding of Zika virus intracellular trafficking and may in future allow for development of novel treatment strategies. Further, we identified a novel mode of action for agents commonly used in studies of virus entry. Schematic representation of differences in ZIKV trafficking in the presence of Baf A1 and NH4Cl.

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Chemical proteomics tracks virus entry and uncovers NCAM1 as Zika virus receptor.

Publications

Chemical proteomics tracks virus entry and uncovers NCAM1 as Zika virus receptor.

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